Method and apparatus for combinatorial chemistry

ABSTRACT

A method and apparatus are provided for performing light-directed reactions in spatially addressable channels within a plurality of channels. One aspect of the invention employs photoactivatable reagents in solutions disposed into spatially addressable flow streams to control the parallel synthesis of molecules immobilized within the channels. The reagents may be photoactivated within a subset of channels at the site of immobilized substrate molecules or at a light-addressable site upstream from the substrate molecules. The method and apparatus of the invention find particularly utility in the synthesis of biopolymer arrays, e.g., oligonucleotides, peptides and carbohydrates, and in the combinatorial synthesis of small molecule arrays for drug discovery.

This application is a division of application Ser. No. 11/559,549, filedNov. 14, 2006, which will issue on Jun. 23, 2009 as U.S. Pat. No.7,550,410, which is a division of U.S. application Ser. No. 09/859,028filed May 16, 2001 now U.S. Pat. No. 7,179,591, issued Feb. 20, 2007,which is a continuation of application Ser. No. 09/305,591, filed May 5,1999, now abandoned.

This invention was made with Government support under Contract No.DE-AC05-96OR22464 awarded by the Department of Energy to Lockheed MartinEnergy Research, Inc., and the Government may have certain rights inthis invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to methods used for performingcombinatorial chemistry and more particularly to a method and apparatusfor the parallel synthesis of chemical arrays using spatiallyaddressable photochemical reaction schemes.

2. Description of the Related Art

Currently there is widespread interest in using combinatorial librariesof oligonucleotides, polypeptides, synthetic oligomers and small organicmolecules to search for biologically active compounds (Kramer, et al.,1993; Houghten, et al., 1992, 1991; Dooley, et al., 1993a-1993b;Eichler, et al., 1993; Pinilla, et al., 1992, 1993). For example,ligands discovered by screening libraries of these types may be usefulin mimicking or blocking natural ligands, or interfering with thenaturally occurring interactions of a biological target. They can alsoprovide a starting point for developing related molecules with moredesirable properties, e.g., higher binding affinity.

Combinatorial libraries of the types useful in this general applicationhave been formed by various solid-phase or solution-phase syntheticmethods. In one approach, beads containing successive precursors to thetarget compounds that form the library are alternately mixed andseparated, with one of a selected number of reagents being added to eachgroup of separated beads at each step (the “split-mix” method: Furka, etal., 1991; Chen et al., 1994; Pham, et al., 1995; Dillard, et al.,1994). Each bead contains only one chemical species, allowing the beadsthemselves to be used for screening. However, the identity of thespecies on each bead must be independently determined. Although severalmethods have been reported for tagging the support beads with moleculesmore readily analyzable than the library members themselves (e.g.,Nestler, et al., 1994; Felder, et al., 1995; Dillard, et al., 1994), theneed for separate identification of each species nonetheless limits theusefulness of this approach for the preparation of very large libraries.

Another general approach involves the synthesis of a combinatoriallibrary as a physically segregated array of compounds (Geysen, et al.,1984, 1985; Southern, 1994; Southern, et al., 1992; Bunin, et al., 1992,1994; DeWitt, et al., 1993). Libraries of compounds have beensynthesized on functionalized resins either coated on (Geysen, et al.,1984, 1985; Bunin, et al., 1992, 1994) or contained within (DeWitt, etal., 1993) arrays of pins, with reactions carried out in separatechambers. Using such an approach, the chemical identity of each libraryelement on the array is associated with an addressable position on thearray. However, in this method, as with the split-mix method,preparation of large libraries would require an undesirably high numberof manipulations and/or a large array of separate reaction vessels orsites.

A method for preparation of potentially high densityposition-addressable arrays on a planar substrate has been reported(Fodor, et al., 1991; Pirrung, et al., 1992). In this method, applicableprimarily to oligomeric compounds, a substrate having photoprotectivegroups is irradiated, using photolithographic mask techniques, inselected regions only, to deprotect surface active groups in thoseselected regions. The entire surface is then treated with a solution ofa selected subunit, which itself has a photoprotected group, to reactthis subunit with the surface groups in the photodeprotected regions.This process is repeated to (i) add a selected subunit at each region ofthe surface, and (ii) build up different-sequence oligomers at known,addressable regions of the surface. This method allows for the synthesisof very large permutation libraries, e.g., 10⁴-10⁶ compounds, in aposition addressable array by parallel subunit addition. For example, inthe case of oligonucleotide libraries, each subunit addition steprequires only four addition reactions, one for each nucleotide added.Thus, in the production of a library of oligonucleotide compounds wheren=8, i.e., a library of 8-mers, 65,536 oligonucleotide compounds can beconstructed with a total of 32 reaction steps (8 subunit additions, 4reactions each).

In cases where the compounds are to be screened for biological activitywhile still attached to the substrate, this method also allows for rapidscreening by binding a reporter-labeled target to the surface anddetermining the positions of bound target. Surface arrays of this typemay be used both for combinatorial library screening (Fodor, et al.,1995; Geysen, et al., 1984, 1985) or for various types ofoligonucleotide analysis, such as sequencing by hybridization (Drmanac,et al., 1993; Southern, 1994; Southern, et al., 1992). However, suchplanar arrays are necessarily limited in the amount (number ofmolecules) of each library species, since the planar region available toeach species is quite small, e.g., on the order of 10²-10³ μm². As aconsequence, the ability to detect binding species on the array may belimited. Further, it is not feasible to carry out solution-phasescreening on a planar array, because of the difficulty of physicallyseparating different array regions carrying different library members.

It would thus be desirable to provide a method and apparatus forpreparing a large combinatorial library of compounds which has theadvantages of (i) parallel synthesis of subunits in known, addressablelibrary positions, (ii) adaptable to virtually any oligomer orsmall-molecule chemistry, (iii) a relatively large area for synthesis ofeach library member, and (iv) screening of individual library compoundsin either solution phase or solid phase. The present invention isdirected to meeting such objectives, and in doing so, overcoming, or atleast reducing the effects of, one or more of the problems set forthabove.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, there is provided amethod for the synthesis of an array of molecules. The moleculessynthesized by this method, e.g., oligonucleotides, peptides,saccharides, small molecules, etc., are immobilized on the inner wallsof a plurality of channels and may have distinct chemical compositionsor sequences within each of the channels. According to one illustrativemethod, a plurality of channels are provided which have immobilized ontheir inner surfaces one or more substrate molecules, the termini of thesubstrate molecules having protecting groups coupled thereto. A subsetof the plurality of channels is exposed to light, for example bydirecting the light through a photolithographic mask, under conditionswhich cause a photochemical reaction within the subset of channeleffective for removing the protecting groups from the substratemolecules. Once the substrate molecules in a subset of the channels aredeprotected in this way, a subunit addition step is performed byproviding within the plurality of channels a desired chemical subunit tobe coupled to the deprotected substrate molecules. By repeating thesesteps, a desired array of molecules is formed within the plurality ofchannels.

The photochemical reaction which occurs in the selected subset ofchannels exposed to light will preferably involve the conversion of aphotoactivatable reagent to a photoactivated reagent, wherein thephotoactivated reagent causes the removal, either directly orindirectly, of the protecting groups at the termini of the substratemolecules. For example, acid precursor compounds, e.g., triarylsulfoniumhexafluoroantimonates, triarylsulfonium hexafluorophosphates,2,1,4-diazonaphthoquinone sulfonates, perhalogenated triazines, and thelike, are well suited for use as photoactivatable compounds according tothis invention. Upon photoactivation within a subset of channels, thephotogenerated acid causes the removal of acid labile protecting groups,e.g., dimethoxytrityl groups, from the substrate molecules.

According to another aspect of the invention, an apparatus is providedfor use in the construction of an array of molecules. The apparatusgenerally comprises a plurality of substantially parallel glass orpolymeric channels, each of said channels having a first end and asecond end. The channels are preferably comprised of substantiallyparallel capillary tubes, or a microchannel array, wherein the channelshave inner diameters in the range of about 1 μm to about 1000 μm. Formost applications, the channel diameters will be in the range of about100 μm to 200 μm, however the apparatus may also be used for making veryhigh-density, closely packed channel arrays with diameters in the rangeof about 1 μm to 10 μm. At least one fluid chamber, generally having oneor more inlet/outlet valves, is continuous with the first and/or secondends of the plurality of channels and is useful for controlling thecomposition and/or flow of solutions into the plurality of channels. Thepresent apparatus further comprises a means for directing light into asubset of the plurality of channels, e.g., by conventionalphotolithographic techniques, for effecting photochemical reactions onlywithin the desired subset of channels.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be best understood by reference to the followingdescription taken in conjunction with the accompanying drawings, inwhich like reference numerals identify like elements, and in which:

FIGS. 1-3 represent partial cross-sectional views of two adjacentchannels in a multichannel array to illustrate one embodiment of theinvention in which a photoactivatable reagent is activated in only asubset of channels in the array to cause the selective deprotection ofthe substrate molecules immobilized within that subset of channels.

FIG. 4 is a cross-sectional view of an illustrative apparatus used inaccordance with the present invention for the synthesis ofthree-dimensional chemical arrays.

FIG. 5 is a representation of selected components, e.g., synthesisblock, synthesis block housing, plurality of channels, etc., of theillustrative apparatus depicted in FIG. 4.

FIG. 6 represents one possible reaction scheme forfunctionalizing/derivatizing the inner walls of the plurality ofchannels in preparation for the synthesis of an array ofoligonucleotides.

FIG. 7 represents one possible reaction scheme for oligonucleotidesynthesis in which the photoactivatable reagent is an acid precursor andthe photoactivated reagent is a photogenerated acid.

While the invention is susceptible to various modifications andalternative forms, specific embodiments thereof have been shown by wayof example in the drawings and are herein described in detail. It shouldbe understood, however, that the description herein of specificembodiments is not intended to limit the invention to the particularforms disclosed, but on the contrary, the intention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the invention as defined by the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

Illustrative embodiments of the invention are described below. In theinterest of clarity, not all features of an actual implementation aredescribed in this specification. It will of course be appreciated thatin the development of any such actual embodiment, numerousimplementation-specific decisions must be made to achieve thedevelopers' specific goals, such as compliance with system-related andbusiness-related constraints, which will vary from one implementation toanother. Moreover, it will be appreciated that such a development effortmight be complex and time-consuming, but would nevertheless be a routineundertaking for those of ordinary skill in the art having the benefit ofthis disclosure.

One illustrative method of the present invention is represented in FIGS.1-3, wherein two adjacent channels 10, 12 in an array of a plurality ofchannels (not shown) are illustrated at selected intermediate stepsduring an array synthesis procedure. Turning to FIG. 1, a solution atleast partly comprised of a soluble photoactivatable reagent 14 isprovided within the channels 10, 12. Immobilized on the inner walls ofthe channels 10, 12 are the substrate molecules being synthesized. Thesubstrate molecules (represented as short lines extending inward fromthe channel walls) will typically have terminal subunits 16 which areprotected by protecting groups 18. Protecting groups 18 serve tosubstantially prevent the terminal subunits 16 from reacting with freesubunits that are present within the channels 10, 12 during arraysynthesis.

An immobilized substrate molecule, as the phrase is used herein, canrefer to a product of the array synthesis process at essentially anystage during array synthesis. Thus, during the array synthesis process,the terminal subunit 16 of the immobilized substrate molecule willtypically represent the chemical subunit most recently added to thesubstrate molecule in a previous step during array synthesis. Forexample, during the synthesis of an oligonucleotide array according tothis invention, the oligonucleotides bound to the inner surfaces of theplurality of channels represent the immobilized substrate molecules.Depending on the array synthesis step being performed, the immobilizedoligonucleotides in the plurality of channels will generally haveterminal subunits comprised of the protected nucleotides added in apreceding subunit addition step. Alternatively, a subset of theplurality of channels may have deprotected nucleotides as their terminalsubunits in preparation for the next subunit addition step. Of course,if the reaction being performed is the first subunit addition step inthe synthesis procedure, the immobilized substrate may be comprised of aconventional linker compound, a derivative thereof, or another compoundsuitably bound on the inner surfaces of the channels for use as animmobilized chemical platform for subsequent array synthesis.

Turning to FIG. 2, a light source 20 is directed selectively within adesired channel or subset of channels by use of a mask 22. In thisexample, the mask 22 allows the light 20 to contact the photoactivatablereagent molecules only in the selected channel 10 but blocks the lightfrom contacting photoactivatable reagent molecules in the other channel12. The light is provided within the channel 10 at an intensity and fora duration effective for causing the conversion of the photoactivatablereagent 14 to photoactivated reagent 24. As a result of light exposurewithin the channel 10, photoactivated reagent 24 is produced. Thephotoactivated reagent 24 then reacts with and causes the removal of theprotecting groups 18 from the terminal subunit 16 of the immobilizedsubstrate molecules. This removal, or deprotection step, provides thedeprotected terminal subunit 32 of the substrate molecule and releasesfree protecting group 34 within the channel 10, as represented in FIG.3.

After performing any desired intermediate steps following deprotectionof the terminal groups of the substrate molecules, (e.g., washingsteps), a subunit addition step is performed by providing within thechannels 10, 12 a solution containing the next desired subunits to bereacted with the deprotected terminal subunits 32. As alluded to above,in this subunit addition step, the subunits that are provided within thechannels during the subunit addition step will generally be protected,i.e., will have protecting groups coupled thereto such that only asingle desired subunit is added to the deprotected terminal subunit 32.Preferably, the subunits that are provided within the channels in thesubunit addition step will have protecting groups coupled thereto of thesame type that was present on the terminal subunit of the substratemolecule prior to the deprotection step. The subunits are provided inthe channels under conditions suitable for reacting with the deprotectedterminal subunits 32 within the channel 10, thereby coupling a newprotected subunit to the growing substrate molecule within that channel.However, the subunit does not react with the terminal subunits of thesubstrate molecules immobilized within the other channel 12 sincedeprotection did not occur in the channel 12. Through repetition ofthese steps, i.e., mask-mediated deprotection in a subset of channelsfollowed by subunit addition of the deprotected substrates, a desiredarray of chemical entities can be synthesized within the plurality ofchannels.

The site-specific application of light to a selected subset of channelsin a multichannel array will typically be achieved using a mask,preferably produced by one or more of the numerous photolithographictechniques that are well known in the art. The source of the lightdirected at the mask and into the desired subset of channels is notcritical, and may vary depending on the particular photochemistryinvolved in the photoactivation step. For many applications, aconventional ultraviolet or visible light source will be suitableprovided it can be directed within the selected subset of channels at asufficient intensity to effect the desired photoactivation reactions. Itshould be noted that other suitable approaches may be used for directinglight into specific channels of the array that do not involve the use ofphotolithographic masks. For example, light may be selectively directedinto the desired subset of channels by a photodiode array having apattern of light point sources. Alternatively, a light beam, e.g., laserbeam, may be scanned over the array such that only the desired subset ofchannels is irradiated. These and other like techniques will be readilyapparent to the skilled individual in this art.

The photoactivatable reagent may be essentially any reagent which, uponlight activated conversion to a photoactivated reagent, undergoes orcauses a desired reaction within the channel(s) in which it has beenactivated. Preferably, the photoactivatable reagent is a reagent thatupon photoactivation reacts with and causes the removal of protectinggroups from the terminal subunits of the immobilized substrate moleculeswithin the channel. Numerous possible photochemical reaction schemes areavailable and will be readily apparent to the skilled individual inlight of this disclosure. These may involve, without limitation,addition reactions, substitution reactions, oxidation reactions,reduction reactions, hydrolysis reactions, and the like. Depending onthe requirements and/or preferences for a particular implementation ofthis embodiment of this invention, suitable photoactivatable reagentsmay be selected such that, upon photoactivation, the reagent isconverted to a desired functionality, e.g., acid, base, thiol reducingagent, alcohol, free radical, or other functionality suitable forreacting with and/or causing the desired removal of protecting groupsfrom the protected terminal subunits of the substrate molecules. Forexample, the following reaction schemes illustrate certain non-limitingexamples of photochemical reactions suitable for converting aphotoactivatable reagent to a photoactivated reagent according to theinvention:

(1) Photogeneration of Acids:

R=H, alkyl, aryl, heteroaryl (e.g., CH.sub.3, CCl.sub.3, CF.sub.3,phenyl)

X=photolabile group (e.g., 2-nitrobenzyl)

(2) Photogeneration of Bases:

R′, R″=H, alkyl, aryl

X=photolabile group (e.g., 2-nitrobenzyloxycarbonyl)

(3) Photogeneration of Thiol Reducing Agents

R=alkyl, hydroxyalkyl (e.g., 2-hydroxyethyl)

X=photolabile group (e.g., 2-nitrobenzyl)

(4) Photogeneration of Alcohols (e.g. for Esterification of ImmobilizedAcids)

R=alkyl, aryl

X=photolabile group (e.g., 2-nitrobenzyl, 2-nitrobenzyloxycarbonyl)

(5) Photogeneration of Free Radicals (e.g., for Additions,Displacements, Etc.)

For example:

The skilled individual will appreciate that there are many types ofphotoaddition and photosubstitution reactions suitable for use in theinvention, not necessarily limited to these specifically describedherein (for example, see Turro, 1978)

In one preferred embodiment of the invention, the photoactivatablereagent is an acid or base precursor. The acid precursor can beconverted to an acid and the base precursor can be converted to a basewithin a channel by a solution photolytic process. In one embodiment,the protecting group on the terminal subunits of the substrate moleculesis acid labile. Thus, the acid precursor is converted to aphotogenerated acid within the subset of channels exposed to light, andthe resulting acid is effective for causing the removal of the acidlabile protecting group from the termini of the substrate moleculeswithin the subset of channels. Accordingly, photoactivatable reagentsmay include, without limitation, 2-naphthol, triarylsulfoniumhexafluoroantimonates, triarylsulfonium hexafluorophosphates,2,1,4-diazonaphthoquinone sulfonates, perhalogenated triazines,N-(2-nitrobenzyloxycarbonyl)piperidine and 2-nitrobenzyl acid esters,derivatives thereof, and other like compounds capable of undergoinglight activated conversion to an acidic or basic form. Many suchcompounds have been reported (see, for example, Gao et al., 1998) andnumerous others will be readily apparent to individuals skilled in theart of photochemistry. Particularly preferred photoactivatable agentsinclude acid compounds, e.g., trichloroacetic acid or trifluoroaceticacid compounds, having photocleavable carboxyl-protecting functions,such as the various α- and phenyl-substituted 2-nitrobenzyl groups,phenacyl groups, benzoinyl groups, arylazidoalkyl groups,2,4-dinitrobenzenesulphenyl groups and N-substituted 2-nitroanilidogroups (see, for example, Pillai, 1987).

The above examples are illustrative only. The selection ofphotoactivatable reagents will of course vary depending on particularchemical entities being synthesized in the array, on the protectinggroups being used in the synthesis strategy, etc. Nonetheless, thechemistries involved in photochemically induced reactions have been welldescribed in the art, and the identification and selection of specificreagents desired for a given implementation can be readily achieved.

The present method may be used in synthesizing a variety ofcombinatorial library types. These may include, for example, arrays ofoligomeric/polymeric compounds, such as different-sequenceoligo/polynucleotides, or analogs thereof, different-sequenceoligo/polypeptides or analogs thereof, position-substitutedoligo/polypeptides, oligo/polysaccharides with different sequences ofsaccharide subunits, lipopeptides/proteins with different permutationsof lipid and/or peptide moieties, glycopeptides/proteins, non-biologicaloligomers/polymers with different sequence permutations, and the like.

A suitable protecting group may include essentially any chemical groupthat is removable, either directly or indirectly, by the chemicalcompound that is photochemically generated within the subset of channelsexposed to light. Typically the protecting groups are comprised ofchemical entities that are substantially labile to acids, bases, thiols,alcohols, free radicals, and/or other functionalities. Numerousprotecting groups will be suitable for use in this invention, e.g.,dimethoxytrityl, 9-fluorenylmethyloxycarbonyl, t-butyloxycarbonyl,methoxymethyl, t-butoxymethyl, siloxymethyl, tetrahydrofuranyl,1-ethoxyethyl, monomethoxytrityl,3-(imidazol-1-ylmethyl)bis(4′,4″-dimethoxyphenyl)-methyl and9-(9-phenyl)xanthenyl, and other like compounds apparent to the skilledindividual (see, for example Greene and Wuts, 1991). The choice ofprotecting group will of course depend on the objectives of theapplication and on the particular photoactivatable reagents being used.For example, in situations where an acid labile protecting group isemployed, such as a dimethoxytrityl group, the photoactivatable reagentthat is used will be one that generates an acid upon exposure to light,wherein the acid is effective for causing the removal of thedimethoxytrityl group.

After the photochemical removal of the protecting groups from theterminal subunits in the subset of channels exposed to light, thechannels may be flushed or otherwise treated as necessary to remove anyundesired products of the photochemical step. Other manipulations mayalso be performed if necessary or desired depending on the specifics ofa given implementation. Subsequently, one or more non-photochemicalreactions, typically referred to herein as subunit addition steps, areperformed by providing in the plurality of channels a solution at leastpartly comprised of one or more of the chemical subunits to be added tothe deprotected substrate. The subunits are provided under conditions inwhich they are chemically coupled to the deprotected termini of thesubstrate molecules in the channels previously exposed to light, but arenot added within the channels which were not exposed to light. Thesubunit added in the subunit addition step may be essentially anychemical entity capable of reacting with and becoming coupled to thedeprotected substrate. In the case where the substrate molecule beingsynthesized is an oligomer/polymer, e.g., an oligonucleotide or peptide,the subunit added will typically be a monomeric unit of thatoligomer/polymer, e.g., a nucleoside or amino acid, respectively.

In one preferred embodiment, the method of the present invention may beused for the synthesis of an oligonucleotide array. The length of theoligonucleotides in the array, i.e., number of nucleotide subunitspresent, is not limiting. In practice, the inner walls of the pluralityof channels within which the array is to be synthesized are suitablyderivatized by any of a number of known techniques so as to provide achemical platform for oligonucleotide synthesis. Methods forderivatizing glass surfaces and other materials for various types ofsolid-phase syntheses are well known (see, for example, U.S. Pat. Nos.5,436,327, 5,142,047, 5,137,765 and 4,992,383).

The photoactivatable reagent provided within the plurality of channelsin this embodiment may be selected from a wide variety of photoreagentsavailable to the skilled artisan. One preferred class ofphotoactivatable reagents is comprised of acid precursor compounds whichcan be converted to acidic species upon exposure to light. For example,2-nitrobenzyl esters, triarylsulfonium hexafluoroantimonates,triarylsulfonium hexafluorophosphates, 2,1,4-diazonaphthoquinonesulfonates, perhalogenated triazines, and other like compounds, can bephotochemically cleaved or activated to produce an acid. Particularlypreferred photoactivatable agents include acid compounds, e.g.,trichloroacetic and trifluoracetic acid compounds, having photocleavablecarboxyl-protecting groups, such as the various α- andphenyl-substituted 2-nitrobenzyl groups, phenacyl groups, benzoinylgroups, arylazidoalkyl groups, 2,4-dinitrobenzenesulphenyl groups andN-substituted 2-nitroanilido groups (see, for example, Pillai, 1987).

Dimethoxytrityl protecting groups are generally preferred in thisembodiment of the invention. Dimethoxytrityl groups are widely used for5′-O protection in conventional oligonucleotide synthesis, and, inaddition, the monomethoxytrityl groups have also been used. A number ofother acid-labile groups have been used for hydroxyl protection in othertypes of syntheses, and are similarly suitable for use in thisinvention. Many of these can be found in Greene and Wuts (1991).Examples of groups that can be cleaved with acetic acids includemethoxymethyl (cleaved with trifluoroacetic acid),t-butoxymethyl(trifluoroacetic acid), siloxymethyl(acetic acid),tetrahydrofuranyl(acetic acid) and 1-ethoxyethyl(acetic acid).

The photoactivatable reagent is provided in the plurality of channelswhich make up the array, and a light source is directed into a desiredsubset of the channels, preferably using a photolithographic mask. Forexample, a mercury arc lamp may be used with spectral filtration toprevent transmission of wavelengths shorter than about 320 nm. Preferredwavelengths for this embodiment are in the range of 320-380 nm,preferably 350-370 nm, since wavelengths shorter than ˜320 nm can damageoligonucleotide molecules and wavelengths longer than ˜380 nm are lessefficient for driving the photoreaction. The light is provided at anintensity and for a duration effective for converting the acid precursorto an acidic form. The photogenerated acid is thereby generated in thelight-exposed channels and causes removal of the dimethoxytrityl orother protecting groups from the terminal nucleotide of an immobilizedoligonucleotide chain. As a result, the terminal nucleotides becomereactive, i.e., available for the addition of a new protected nucleosidemonomer at their termini. Thus, by providing a protected nucleosidemonomer within the plurality of channels, the detritylated termini reactwith the new protected nucleoside monomer and add it to the growingoligonucleotide chain, while the oligonucleotide chains having terminalnucleotides that were not detritylated (i.e., those that were notexposed to photogenerated acid) do not add the new protected nucleosidemonomer. Immobilized oligonucleotide chains in a different subset ofchannels may then be detritylated using a different photolithographicmask, followed by the addition of a different protected nucleosidemonomer. These basic steps may be repeated until a desired array ofdifferent-sequence oligonucleotides are synthesized in the plurality ofchannels.

Alternative chemistries for the phosphite-triester andphosphate-triester synthesis of oligonucleotides are well known and canalso be used in the array synthesis processes described herein.Moreover, the synthesis of arrays of RNA oligomers may be carried outsimilarly, for example, using analogous ribonucleotide monomers with2′-O protecting groups.

In other embodiments of the invention, photochemically activatedreagents may be used to block functional groups of immobilizedsubstrates in specific channels so that they remain unaltered duringsubsequent reactions of nonprotected substrates in other channels.Photochemical reactions may also be used to indirectly control blockingof other types of reactions. For example, the protection of primaryamines by citraconic anhydride (Dixon and Perham, 1968) may becontrolled by altering the pH of the reacting solution. The reaction ofthe anhydride with an amine to produce an unreactive amide proceeds atpH 8 but is reversible at pH 4. Therefore, photolysis of a photolabileester to produce an acid to lower the pH of the anhydride solution willprevent the blocking reaction, whereas photolysis of a photolabilecarbamate to produce a hindered secondary amine could be used to promotethe reaction by increasing the pH of the solution. These and otherexamples will be readily apparent to the skilled individual in light ofthis disclosure.

It will be apparent that the specific example described herein isnon-limiting and many variations to the method of this invention will beapparent to the skilled individual. For example, as described above, thecombinatorial synthesis strategy may be designed such that the subunitaddition step provides a chemical species that reacts only with theimmobilized substrates which underwent the photochemically inducedremoval of protecting group. Alternatively, the synthesis strategy maybe designed such that the added subunit reacts only in those channelswherein the terminal subunits did not undergo deprotection.

The present invention further provides an apparatus useful for producingcombinatorial chemical arrays, e.g., for the production of polymerarrays such as oligonucleotide arrays and oligopeptide arrays. Anexemplary apparatus is shown schematically in FIG. 4 and several of thecore components of the apparatus are further represented in FIG. 5. Asynthesis block 41 containing a plurality of parallel channels 42 isenclosed in a synthesis block housing 43. For simplicity ofillustration, the synthesis block is shown here with a low density(5.times.6) array of widely-spaced, large-diameter channels.

The channels which make up the desired array may be formed by any of avariety of approaches and may take numerous forms and/or geometries. Inone illustrative embodiment, the channels are comprised of a pluralityof capillary tubes, e.g., those known in the art for performingcapillary electrophoresis, arranged in a highly dense array.Alternatively, the plurality of channels may be comprised of amicrochannel array. Although the number, density and specificarrangement of channels in the array is not limiting, the arrays willtypically contain greater than 100 channels, more typically greater than1000 channels, and sometimes greater than 10,000 or more channels. Theinner diameters of the channels will be less than about 1 mm in mostinstances, however larger channel diameters will also be useful for someapplications. Typically, the channels will be cylindrical, i.e., willhave a substantially circular cross-section, and will have innerdiameters in the range of about 1 to 500 μm, preferably in the range ofabout 1 to 100 μm. In addition, high density, closely packed arrayshaving channel diameters in the range of about 1 to 10 μm are alsopossible in accordance with this invention.

The synthesis block 41 may be made from any of a variety of materials,e.g., glass, polymer, or other suitable materials, depending on thespecific application for which the completed array is to be used. Forexample, a synthesis block made of a polymer material may be preferredwhere the block is to be cut into thin transverse sections. The innerwalls of the synthesis channels 42 are generally chemically modified orotherwise derivatized prior to array synthesis by covalent attachment ofsuitable linker moieties which serve as the chemical platform forsolid-phase synthesis within the channels. For example, the inner wallsof the channels 42 may be treated to form chemically reactive surfacegroups, such as carboxyl, hydroxyl, or amine groups on the inner wallportion and the reactive groups then chemically reacted with a protectedlinker moiety to which the first subunit may be added at the beginningof array synthesis. Methods for derivatizing glass surfaces for varioustypes of solid-phase syntheses are well known (see, for example, U.S.Pat. Nos. 5,436,327, 5,142,047, 5,137,765 and 4,992,383). In oneillustrative example, a linker arm will contain a hydroxyl group whichis protected as the dimethoxytrityl ether. Deprotection, i.e., removalof the limethoxytrityl protecting group, in a specific subset ofchannels, therefore allows coupling to a first chemical subunit, e.g.,the first nucleoside monomer during oligonucleotide array synthesis.

The synthesis block housing 43 is typically made of metal, such asstainless steel, but may be made of any substantially rigid material,including Teflon or other chemically inert plastics. A gasket 44,preferably made of Teflon, silicon rubber or other chemically inertmaterial, separates one end of the synthesis block 41 and synthesisblock housing 43 from a transparent plate 45 to create a fluid chamber46. The fluid chamber 46 is continuous with the ends of the plurality ofchannels, and is thereby capable of providing a desired solution withinthe channels. The gasket 44 further provides a seal to prevent fluidleakage between the sides of the housing and synthesis block. The plate45 is held in place by an end cap 47 which is separated from the plateby a second gasket 48. The end cap may be made of the same material asthe synthesis block housing, or another suitable material.

A photolithographic mask 49 is positioned above the transparent platesuch that the ends of specific channels may be exposed to light whichpasses through transparent regions 50 in the mask 49 and thereforethrough the transparent plate or fluid chamber. Radiation from a lightsource 51, such as a mercury arc lamp or laser, may be spectrallyfiltered, focused and collimated as necessary to produce essentiallymonochromatic, collimated light of the desired wavelength. Forhigh-density arrays of channels, the thicknesses of the transparentplate 45 and fluid chamber 46 are preferably minimized to reducespreading of the light due to refraction. It may be suitable to usemicrofabricated arrays of lenses in place of the transparent plate tofocus the light on the channels, thereby avoiding light spreading tononselected channels. Furthermore, one could expel the liquid from thetop chamber 46 and bring the top of the synthesis block into directcontact with the transparent plate 45 during the photoexposure. However,this would not allow continuous flow of reagent into the synthesis blockduring exposure. Holes in the transparent plate 45, gasket 48 and endcap 47 are aligned to create a first passageway 52 through which fluidsmay be added to the fluid chamber 46 and a second passageway 53 throughwhich fluids may flow out of the chamber. The flow of fluids into andout of the chamber is controlled by valves 54 and 55, respectively,which may be connected to the passageways by means of tubing 65. A valve55 may be further connected by means of tubing to a waste receptacle. Atthe opposite end of the synthesis assembly a second fluid chamber 56 iscreated by the end plate 57, which is separated from the end of thesynthesis block and synthesis block housing by a gasket 58 and held inplace by a second end cap 59 and gasket 60. The end plate may be made ofglass, metal or any other substantially rigid and chemically inertmaterial. Holes in the end plate 57, the second end cap 59, and thegasket 60, are aligned to create a first passageway 61 for the additionof fluids to the chamber 56 and second passageway 62 through whichfluids may flow out of the chamber. The flow of fluids throughpassageways 61 and 62 is controlled by valves 63 and 64, respectively,which are connected to passageways by means of tubing to a pump or otherdevice for adding solvents to the fluid chamber, 56, and valve 64 isconnected by means of tubing to a waste receptacle. The synthesis blockassembly may be held together by screws or other fasteners (not shown)which connect the end cap 47 to the second end cap 59.

With further reference to FIG. 4, an illustrative combinatorial arraysynthesis may be carried out in the disclosed apparatus as follows. Asolution of photoactivatable reagent is added to the fluid chamber 46 byopening valves 53 and 54 and by use of a pump, syringe or other means ofapplying hydraulic pressure to the solution (not shown). After thechamber 46 is filled with reagent solution, valve 54 is closed and valve63 is opened to cause the reagent to flow through the channels of thesynthesis block. Light is then directed into the ends of a selectedsubset of channels in the synthesis block via mask 49 to cause thephotoactivation of a photoactivatable reagent within the subset ofchannels. The solution flow may be discontinued prior to photoactivationin cases where the light is able to pass through to entire length of thechannel or a major portion thereof, thereby causing photoactivation ofreagent throughout the major portion of the channel. Alternatively, incases where the light is substantially absorbed in a portion of thechannel proximal to the light source, the flow of reagent solution maybe continued during all or part of the photoactivation step in order todistribute the photoactivated reagent throughout the length of thechannel.

In order to prevent the buildup of photoactivated reagent in fluidchamber 46 which could enter non-selected channels, valve 55 may bepartially or intermittently opened in order to add fresh reagentsolution to the chamber and to flush photoactivated reagent out of thechamber. Other means will also be suitable to prevent the undesiredentry of photoactivated reagent in non-selected channels. For example,following the addition of reagent solution to the synthesis channels 42and prior to the photoactivation step, the reagent solution can beflushed out of the chamber 46 with solvent which contains nophotoactivatable reagent by opening one valve 55 and closing the othervalve 63. The valve 63 may be used for flushing reagents from the bottomof fluid chamber 56 and/or for backflushing the synthesis block withsolvents. The flushing step will also remove reagent solution from asmall portion of the channel proximal to the fluid chamber.

The selected subset of channels are then exposed to light through themask 49. Because no reagent is present in the fluid chamber, nophotoactivated reagent can be formed in the chamber and enternon-selected channels. The steps of reagent addition, solvent flushingand photoactivation can be repeated, if necessary, until the entirelength of the selected channels are exposed to photoactivated reagent.Following reaction of photoactivated reagent in the subset of selectedchannels, a subunit addition step is performed in which a chemicalsubunit or other moiety is provided in the plurality of channels. Thesubunit reacts only with substrate molecules within the subset ofchannels where the photoactivated reagent was generated. A differentsubset of channels are then selected for reaction with a photoactivatedreagent using a different photolithographic mask, and these steps arerepeated until the desired array of chemical entities has beensynthesized in the plurality of channels. Of course, intermediatenonphotochemical reaction steps may also be included in the totalsynthesis, as necessary and/or desired.

It will be apparent that there are many variations in the design andoperation of the apparatus described above. The fundamental requirementsare: (1) a means of controlling the flow of reagents and solventsthrough the array of channels within the synthesis block, such as amicrochannel plate or capillary array, and (2) a means of directinglight into specific channels or groups of channels of the synthesisblock. Other means of performing these steps may include, for example,the application of electrical voltages to the solutions in fluidchambers 46 and 56 to cause electrokinetic flow through the synthesisblock channels, and the use of a focused laser beam to sequentiallyphotoactivate reagents in selected channels by either rastering thesynthesis block assembly or scanning the laser beam.

In one illustrative embodiment of the invention, following synthesis ofthe desired array of chemical entities, the three-dimensionalmultichannel structure may be sliced into multiple two-dimensionalarrays. The total surface area occupied by each chemical entity in a‘two-dimensional’ array of this type is given by: Area=πdh, where d isthe diameter of the channel and h is the height (thickness) of theslice. Thus, the area for each member of the array in a slice 10 μmthick with 10 μm diameter channels is 314 μm², whereas the area for eachmember of a conventional planar array containing 10 μm diameter spots isonly 78.5 μm², or one-fourth of the area in the 10 μm thick slices. Forligand-binding applications, such as the use of oligonucleotide arraysfor hybridization analysis of nucleic acids, the increased probedensities per 10 μm array member results in improved detection of boundtarget molecules. A synthesis block could potentially yield 1000 10-μmthick arrays per cm of its original length. The sliced sections willpreferably have thicknesses in the range of about 1 μm to about 100 μm,more preferably in the range of about 1 μm to about 10 μm, however,thicker array slices, e.g., greater than 100 μm, could also be used forflow through applications such as those described by Beattie et al.Synthesis blocks made of silicon or glass may be sectioned by methodsused in the industry for the preparation of silicon wafers ormultichannel plates, with precautions to avoid the generation of hightemperatures in the processed material. Synthesis blocks made of organicpolymers, glass capillaries, composite materials, and the like, may besliced into thin sections using microtomes or similar devices forslicing such materials. The thin sections may be mounted on slides fordurability and ease of handling.

In situations where relatively short channels are used, or where thephotoactivated reagents are transparent to the activating wavelengths oflight, it may be desired to activate the photoactivatable reagentthrough essentially the entire length of the channel, e.g., byilluminating a static solution from one end of the channel. In thiscase, the activating light is able to pass through the entire length ofthe channel either due to low intrinsic absorbance and/or concentrationof the activatable reagent and its photoproducts, or because thephotoreaction products are nonabsorbant and allow the light to penetratefurther into the solution as the reaction proceeds. For longer channels,or in cases of high reagent or photoproduct absorbance, the reagent maybe photoactivated in a portion of the channel proximal to the lightsource and the activated reagent moved through the length of the channelby fluid transport to the distal end. The reagent flow andphotoactivation may be continuous or intermittent until the reactionwith the substrate is completed throughout the channel.

In a further embodiment of the invention, the method and/or apparatusdescribed herein may be used to create arrays of biomolecules byactivating linker groups in specific channels followed by flowingsolutions of desired molecules through the channel block. For example,linker groups bearing a primary amine protected as themonomethyoxytrityl derivative may be deprotected by photogeneration ofan acid in a specific channel or channels. Reaction of the deprotectedamines with a cross-linking agent, such as glutaraldehyde, would allowattachment of a biomolecule containing a primary amine, e.g., a proteinor amino-modified nucleic acid. The biomolecule would attach only tothose channels which were initially deprotected. Such steps could berepeated to attach different biomolecules within different channelsuntil a desired array was completed.

Although the embodiments described herein will most typically be used inthe fabrication of high-density arrays using individual channels havingsub-millimeter diameters, arrays channels having considerably largerdiameters could be used for the synthesis of greater quantities ofindividual products. It may be beneficial to fill such channels with asolid support, such as silica or polymeric microbeads, to increase thesynthesis area and product yield. If desired, the photoreactions may beperformed in an area of the channel which does not contain the solidsupport material to avoid potential light blockage by the solid support.

The illustrative apparatus is also useful for performing photochemicalreactions on a variety of immobilized substrates within the channels.Light directed into individual channels, e.g., by photolithographic orother means, may be propagated through the channel cavity or channelwall by internal reflection. In this way, photochemical reactions maytake place on molecules immobilized on the interior surface of thechannel by an evanescent wave effect. If desired, the channel cavitycould be continuously flushed with a nonabsorbing solvent to removereleased photolysis by-products. Capillary arrays made, for example,from tubing having an outer surface or layer with an index of refractionlower than that of the inner wall of the tubing could be used for totalinternal reflection within the inner wall and to prevent light fromleaking between adjacent capillaries. Similarly, the use of solvents orsolutions with higher refractive indices than the capillary walls couldbe used to confine the light to the liquid filled channel cavity.Photochemical reactions in immobilized molecules by direct exposure tolight as well as indirect photochemical reactions caused byphotoactivation of soluble reagents may therefore be used in the totalsynthesis of desired compounds in three-dimensional arrays of channels.

The following examples are provided to demonstrate certain illustrativeembodiments of this invention. It should be appreciated by those skilledin the art that the techniques disclosed in the examples which followrepresent those found by the inventors to function in the practice ofthe invention and thus can be considered to constitute examples ofparticular modes for its practice. However, those skilled in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

EXAMPLE Example 1 Light-Induced Deprotection ofDimethoxytrityl-Protected Nucleosides

A solution containing 5′-O-dimethoxytrityl thymidine (1 mM) and2-naphthol (10 mM) in methylene chloride was placed in a quartz cuvettehaving a 2 mm path length and exposed to light from a 200 Whigh-pressure mercury arc lamp for 10 minutes. The light was passedthrough bandpass and sharpcut filters to give maximum transmission at˜360 nm wavelength with a cutoff at ˜320 nm. The irradiated solution wasanalyzed by high performance liquid chromatography (HPLC) and comparedwith a non-irradiated solution stored in the dark for the same period oftime. The analyses showed essentially complete (97%) detritylation ofthe exposed nucleoside to give thymidine and no detritylation of thenon-exposed sample. 2-Naphthol is known to be substantially more acidicin the excited state (SI, pK=3.1) than in the ground state (So, pK=9.5)(Calvert and Pitts, 1966). The efficiency of this reaction is somewhatsurprising in view of the short lifetime of the S1 excited state (˜10nanoseconds) and the fact that the triplet (T1) state is not acidic(pK˜8). A similar experiment was performed using 4-nitrophenol (So,pK=7.15) instead of 2-naphthol. 4-nitrophenol absorbs strongly at longerwavelengths than does 2-naphthol and was exposed using filters for 365nm maximum transmission with a 345 nm cutoff. Although detritylation of5′-O-dimethoxytrityl thymidine in the exposed solution proceeded moreslowly with this phenol, it continued in the dark for several minutesafter the exposure, indicating the formation of a long-lived acidicspecies.

Example 2 Oligonucleotide Array Synthesis

A synthesis block containing parallel glass capillary channelsapproximately 50 μm in diameter and approximately 1 cm in length is usedin this example. The inner walls of the channels are first derivatizedwith linkers bearing a dimethoxytrityl ether which can be deprotectedwith a photogenerated acid to produce an hydroxyl group for attachmentof the first nucleotide. The method for functionalizing the channelwalls is shown in FIG. 6. The capillary surfaces are first treated with3-glycidoxypropyltrimethoxysilane 61 (2% in ethanol). Functionalizationof glass surfaces with this reagent is well known. After rinsing thecapillaries with ethanol and drying them via purging with inert gas(e.g. nitrogen or argon) the silane coating is allowed to cure at roomtemperature for several hours or, alternatively, the synthesis block isremoved from the apparatus and heated (e.g., at 110° C. for 5-10minutes) to cure the silane layer. The linker glycidyl groups are thenreacted with O-(4,4′-dimethoxy-trityl)ethanolamine 62 to give thedimethoxytrityl ether-terminated linker 63. The secondary hydroxyl andamino groups in the linker molecule can be cyclized by reaction withdiethyl carbonate or carbonyl chloride to give the fully protectedlinker 64.

Dimethoxytrityl (DMT)-protected linkers in selected channels aredeblocked by adding a solution of photoactivatable acid to all channelsand photogenerating the acid in selected channels by illuminationthrough a mask, as described elsewhere in this application. Thedetritylation reaction is shown in FIG. 7. A 1-10% (w/v) solution of1-[2-nitrophenyl]ethyl-1-trichloroacetate 75 in methylene chloride isprovided within the channels and illumination of this reagent withultraviolet light causes its dissociation into trichloroacetic acid 76and o-nitrosoacetophenone 77. Irradiation in the subset of channelsdefined/exposed by the mask is provided at an intensity of about ˜10-30mW/cm² for 5-30 minutes depending on reagent concentration and lightintensity to give ˜50-100% deprotection of the acid. The light source isa mercury lamp which has a spectral filter to prevent transmission oflight shorter than about 320 nm. The acid that is generated within thesubset of channels causes detritylation of the DMT ether to produce aterminal hydroxyl group on the linker 78. Following deprotection of thehydroxyl groups in the selected subset of channels, a acetonitrilesolution of oligonucleotide synthesis monomer having a 5′-O-DMTprotecting group is added to the plurality of channels in the presenceof a phosphoramidite activator (e.g., tetrazole) for coupling theprotected monomer to the deprotected linkers. Preferred protectedmonomers for deoxyribonucleic acid synthesis are5′-O-(4,4′-dimethoxytrityl)-deoxynucleoside-3′-O—(N,N-diisopropylamino-O-cyanoethyl)phosphoramidites79 in which the exocyclic amine groups are protected as the4-nitro-phenylethoxycarbonyl (NPEOC) derivatives (see, for example,Himmelsbach, 1984). The linker groups in a second set of channels isthen deprotected by the above approach and a different monomer is added.These steps are repeated until the linkers of all channels have beenreacted with a first monomer.

The steps of monomer addition, capping and phosphite oxidation arecarried out using standard reagents and methods for the phosphoramiditesynthesis method. The DMT protected 5′-O nucleotide terminus in selectedchannels is then deprotected with photogenerated acid as in the linkerdeprotection step. The synthesis steps of monomer addition anddeprotection with photogenerated acid are continued using differentcombinations of masks until all desired sequences in the array arecompleted. Final deprotection of the exocyclic amines and phosphates iscarried by treatment with 0.5 M DBU in dry pyridine for 30-60 minutes atroom temperature to remove the NPEOC and cyanoethyl groups withoutcleavage of the linker.

The particular embodiments disclosed above are illustrative only, as theinvention may be modified and practiced in different but equivalentmanners apparent to those skilled in the art having the benefit of theteachings herein. More specifically, it will be apparent that certaincompounds that are chemically, structurally and/or functionally relatedto those disclosed herein may be substituted in the methods of thisinvention while the same or similar results would be achieved.Furthermore, no limitations are intended to the details of constructionor design herein shown, other than as described in the claims below. Itis therefore evident that the particular embodiments disclosed above maybe altered or modified and all such variations are considered within thescope and spirit of the invention. Accordingly, the protection soughtherein is as set forth in the claims below.

REFERENCES

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1. A method for the synthesis of oligonucleotides comprising the stepsof: a) contacting an oligonucleotide with a solution comprising acarboxylic acid precursor selected from the group consisting of aceticacids, haloacetic acids, and mixtures thereof; wherein a terminus of theoligonucleotide is protected with an acid-labile protecting group; andwherein a carboxyl group of the acid precursor is protected with aphotolabile protecting group; b) exposing the solution to a radiationsource sufficient to cause deprotection of the carboxylic acid precursorto provide an acid compound for removing the acid-labile terminalprotecting group of the oligonucleotide; c) adding a nucleotide subunitto the deprotected terminus of the oligonucleotide; wherein a terminusof the added subunit is also protected with an acid-labile protectinggroup.
 2. A method for the synthesis of oligonucleotides comprising thesteps of: a) exposing a precursor solution comprising a carboxylic acidprecursor selected from the group consisting of acetic acids, haloaceticacids, and mixtures thereof, wherein a carboxyl group of the acidprecursor is protected with a photolabile protecting group, to aradiation source sufficient to cause deprotection of the carboxyl group;b) contacting an oligonucleotide having an acid-labile protecting groupat a terminus, with the irradiated precursor solution for a period oftime sufficient to cause deprotection of the oligonucleotide terminus;c) adding a nucleotide subunit to the deprotected terminus of theoligonucleotide; wherein a terminus of the added subunit is alsoprotected with an acid-labile protecting group.
 3. A method for thesynthesis of oligonucleotides comprising the steps of: a) contacting anoligonucleotide with an acid precursor solution comprising a phenoliccompound; wherein a terminus of the oligonucleotide is protected with anacid-labile protecting group; b) exposing the precursor solution to aradiation source sufficient to cause increased acidity of the precursorsolution for deprotecting the terminus of the oligonucleotide; c) addinga nucleotide subunit to the deprotected terminus of the oligonucleotide;wherein a terminus of the added subunit is also protected with anacid-labile protecting group; and d) optionally, repeating synthesissteps comprising at least steps a-c until the desired oligonucleotidehas been synthesized.
 4. A method for the synthesis of oligonucleotidescomprising the steps of: a) exposing a precursor solution comprising aphenolic compound to a radiation source sufficient to increase anacidity of the solution sufficient for removing an acid-labileprotecting group from a terminus of an oligonucleotide; b) contacting anoligonucleotide having an acid-labile protecting group at the terminus,with the irradiated precursor solution for a period of time sufficientto cause deprotection of the terminus of the oligonucleotide; c) addinga nucleotide subunit to the deprotected terminus of the oligonucleotide;wherein a terminus of the added subunit is also protected with anacid-labile protecting group; and d) optionally, repeating synthesissteps comprising at least steps a-c until the desired oligonucleotidehas been synthesized.
 5. The method of claim 1, wherein the haloaceticacids are selected from the group consisting of dichloroacetic acids,trichloroacetic acids, trifluoroacetic acids, and mixtures thereof. 6.The method of claim 1, wherein the photolabile protecting group isselected from the group consisting of 2-nitrobenzyl groups, α- andphenyl-substituted 2-nitrobenzyl groups, phenacyl groups, benzoinylgroups, and 2,4-dinitrobenzenesulphenyl groups.
 7. The method of claim1, wherein the acid-labile protecting group of the oligonucleotide isselected from the group consisting of dimethoxytrityl,monomethoxytrityl,3-(imidazol-1-ylmethyl)bis(4′,4″-dimethoxyphenyl)methyl and9-(9-phenyl)xanthenyl.
 8. The method of claim 2, wherein the haloaceticacids are selected from the group consisting of dichloroacetic acid,trichloroacetic acid, trifluoroacetic acid, and mixtures thereof.
 9. Themethod of claim 2, wherein the photolabile protecting group is selectedfrom the group consisting of 2-nitrobenzyl groups, α- andphenyl-substituted 2-nitrobenzyl groups, phenacyl groups, benzoinylgroups, and 2,4-dinitrobenzenesulphenyl groups.
 10. The method of claim2, wherein the acid-labile protecting group of the oligonucleotide isselected from the group consisting of dimethoxytrityl,monomethoxytrityl,3-(imidazol-1-ylmethyl)bis(4′,4″-dimethoxyphenyl)methyl and9-(9-phenyl)xanthenyl.
 11. The method of claim 3, wherein the phenoliccompound is selected from the group consisting of 2-naphthol,4-nitrophenol, and mixtures thereof.
 12. The method of claim 3, whereinthe acid-labile protecting group of the oligonucleotide is selected fromthe group consisting of dimethoxytrityl, monomethoxytrityl,3-(imidazol-1-ylmethyl)bis(4′,4″-dimethoxyphenyl)methyl and9-(9-phenyl)xanthenyl.
 13. The method of claim 4, wherein the phenoliccompound is selected from the group consisting of 2-naphthol,4-nitrophenol, and mixtures thereof.
 14. The method of claim 4, whereinthe acid-labile protecting group of the oligonucleotide is selected fromthe group consisting of dimethoxytrityl, monomethoxytrityl,3-(imidazol-1-ylmethyl)bis(4′,4″-dimethoxyphenyl)methyl and9-(9-phenyl)xanthenyl.
 15. The method of claim 1, wherein theacid-labile protecting group of the nucleotide subunit is selected fromthe group consisting of dimethoxytrityl, monomethoxytrityl,3-(imidazol-1-ylmethyl)bis(4′,4″-dimethoxyphenyl)methyl and9-(9-phenyl)xanthenyl.
 16. The method of claim 2, wherein theacid-labile protecting group of the nucleotide subunit is selected fromthe group consisting of dimethoxytrityl, monomethoxytrityl,3-(imidazol-1-ylmethyl)bis(4′,4″-dimethoxyphenyl)methyl and9-(9-phenyl)xanthenyl.
 17. The method of claim 3, wherein theacid-labile protecting group of the nucleotide subunit is selected fromthe group consisting of dimethoxytrityl, monomethoxytrityl,3-(imidazol-1-ylmethyl)bis(4′,4″-dimethoxyphenyl)methyl and9-(9-phenyl)xanthenyl.
 18. The method of claim 4, wherein theacid-labile protecting group of the nucleotide subunit is selected fromthe group consisting of dimethoxytrityl, monomethoxytrityl,3-(imidazol-1-ylmethyl)bis(4′,4″-dimethoxyphenyl)methyl and9-(9-phenyl)xanthenyl.
 19. The method of claim 1, further comprisingrepeating synthesis steps comprising at least steps a-c until thedesired oligonucleotide has been synthesized.
 20. The method of claim 2,further comprising repeating synthesis steps comprising at least stepsa-c until the desired oligonucleotide has been synthesized.